Establishment Of Conditionally Reprogrammed Human Normal Airway Epith Elial Cells And Influenza Virus H1N1 Infection Model

H1N1 Influenza virus,influenza A(H1N1)pdm09,is the member of orthomyxoviridae RNA viruses.Influenza A(H1N1)pdm09 can infect humans,pigs and other vertebrates.It caused 2009 influenza pandemic,and also the world's pandemic in 1918,1957 and 1968,resulting in severe social and public health problems.Different from the seasonal influenza,influenza A(H1N1)pdm09 replicates in the lower respiratory tract and may lead to bacterial and viral pneumonia,respiratory failure and even death.H1N1 influenza virus genome is a multi-segment single-stranded RNA virus and prone to recombination mutation and antigen drift.This feature of influenza A(H1N1)pdm09 greatly limits the efficacy of influenza vaccine and anti-viral drug.Therefore,it is of great importance to have an influenza infection model which is more close to the physiological conditions in vitro.Cell line models or animal models have been used for H1N1 influenza virus studies.There exists the species difference between human and animal.The common used cell lines,such as A549 lung cancer cell line,16HBE(oncogene immortalized cell line),FRhL,MRC-5,WI-38 or PER.C6 cell lines,are either cancer cell lines or viral/oncogene immortalized cell lines.These cell lines usually lost the normal physiological functions due to changed genetic background and phenotypes.Human airway mucosa is the original target of H1N1 infection and the first line of defense against H1N1.Establishment of human normal airway epithelial cell model is the natural physiological infection model to study the immediate interaction between H1N1 and the host cells.In this study,we established the human normal tracheal epithelial cells(HNTEC)and the normal human pulmonary epithelial cells(HNPEC)by conditional reprogramming(CR)techniquewithout transduction of exogenous genes.These two cell lines propagated rapidly in culture.The short tandem repeat(STR)analysis show that genotypeof HNTEC is AMEL/X/X,D3S1358/15/17,D13S317/8/14,D7S820/9/12,D16S539/11/14,Penta E/11,D2S441/12/14,TPOX/8,TH01/6/9,D2S1338/21/24,CSF1P0/11/12,Penta D/8/11,D10S1248/13/15,D19S433/14/15.2,VWA/18/19,D21S11/30/31,D18S51/14,D6S1043/12/18,D8S1179/10/13,D5S818/11,D12S391 1 19/24,FGA/23 and genotype of HNPEC is AMEL/X/X,D3S1358/16,D13S317/8/9,D7S820/11,D16S539/11/12,Penta E/14/16,D2S441/10/12,TPOX/8/11 D01S1248/14/15,D19S433/13/14.2,vWA/14/16,D21S11/30,D18S51/13/24,CSF1PO/12/13,Penta D/9/11,D01S1248/14/15,D6S1043/11,D8S1179/10/15,D5S818/12,D12S391/15/25,FGA/22/25.The STR results indicate that these two cell lines were newly established without matching any other cell lines registered or published before.The karyotype analysis confirmed that the two cell lines have the normal diploid karyotypes without any chromosome rearrangment or translocation.The soft agar experiment showed that HNTEC and HNPEC cells possess low colony formation efficiency which is different from lung cancer cells A549.In addition,HNTEC and HNPEC cells grew asthe clear boundary and smooth spheroids in matrigel 3D culture in vitro while lung cancer cells A549 formednon-structured aggregates.DNA damage experiments also showed that these two cell lines have the normal p53-p21signaling pathways.The immunofluorescence staining by specific markers indicated that HNTEC and HNPEC cells express the tracheal and pulmonary specific protein cytokeratin 5(CK5),epithelial cell specific protein cytokeratin 14(CK14),and the sternness marker p63.These results demonstrated that these two cell lines are human normal airway epithelial cellsNext,we investigated the sensitivity and tropism of HNTEC and HNPEC cells to H1N1 infection.We inoculated influenza A(H1N1)pdm09with MOI of 0.001 and observed the cytopathic effect(CPE)at 0 h,12h,24h,48h and 72h post infection(p.i.).The CPE of the host cells developed gradually after H1N1 infection in a time-depended manner.The morphologic changes of cells include rounding,shrinking and detaching.More importantly,the CPE of HNPEC cells was observed earlier than that of HNTEC cells.The pulmonary originated HNPEC cells are more sensitive than tracheal originated HNTEC cells.The proliferation capacity of H1N1 influenza virus in different cell lines are in following order:HNPEC,HNTEC,A549,16HBE.In accordance with this observation,we obtained the same result by immunofluorescence using specific antibody against H1N1 pdm09envelope protein.The viral protein was observed as early as 24 h p.i.in HNPEC cells.The indensityand quantity of the viral protein are the highest in HNPEC cells among these infected cells at 72 h p.i..In HNTEC cells,the viral protein was also observed at 24 h p.i.and increased obviously at 72 h p.i..In contrast,the viral protein was observed at 48 h p.i.in A549 cells.The immortalized 16HBE cells are not sensitive to H1N1 infection.These results demonstrated that H1N1 pdm09 is prone to replicate in pulmonary cells resulting in lower airway infection and severe respiratory diseases thereafter.Lastly,we tried to reconstruct a polarized tracheal epithelium 3D model in vitro by air-liquid interface(ALI)culture.This 3D pseudostratified mucosal epithelium is very similar to its originated tracheal tissue.In summary,we successfully establish the normal upper and lower airway epithelial cell lines and H1N1 infection model in vitro.Our study may provide a novel influenza infection model which is more close to the physiological conditions in vitro.